Quantification of Membrane Proteins Using Nonspecific Protease

Oct 21, 2009 - Department of Cell & Organism Biology, Lund University. , ∥ .... Marianne Sandin , Morten Krogh , Marie Ovenberger , Erik Fredlund , ...
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Quantification of Membrane Proteins Using Nonspecific Protease Digestions Maria Bendz,*,†,‡ Mirja Carlsson Mo ¨ ller,§ Giorgio Arrigoni,†,| Åsa Wa˚hlander,† Roberto Stella,| †,⊥ Salvatore Cappadona, Fredrik Levander,† Lars Hederstedt,§ and Peter James*,† Protein Technology, Department of Immunotechnology, CREATE Health, Lund University, Sweden, Centre for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, Sweden, Department of Cell & Organism Biology, Lund University, Sweden, Department of Biological Chemistry, University of Padova, Italy, and Department of Bioengineering, IIT Unit, Politecnico di Milano, Italy. Received August 20, 2009

Abstract: We present a mass spectrometry-based method for the identification and quantification of membrane proteins using the low-specificity protease Proteinase K, at very high pH, to digest proteins isolated by a modified SDS-PAGE protocol. The resulting peptides are modified with a fragmentation-directing isotope labeled tag. We apply the method to quantify differences in membrane protein expression of Bacillus subtilis grown in the presence or absence of glucose. Keywords: membrane proteins • proteinase K • N-terminal labeling • relative quantification • Bacillus subtilis

Introduction Approximately 25% of all translated genes code for membrane proteins (defined as possessing two or more transmembrane domains).1 This number can increase dramatically, up to 40%, if one includes membrane-anchored proteins and those that have a single trans-membrane (TM) helix or are lipidanchored. These estimates of the number of membrane proteins are based on the prediction of R-helical membrane domains and do not include beta-barrel proteins that are found in prokaryotes and eukaryotic organelles where they can account for 2% of the genes.2 Despite advances in protein technology, membrane proteins still represent one of the most difficult classes of proteins to study. The majority of proteins that are generally identified are either membrane associated or have large extra-membrane domains that behave like soluble proteins. There are three main reasons for this: the separation problems associated with the hydrophobicity of the proteins or peptides, the refractory nature toward digestion, and the large average size of peptides produced by specific endoproteases. Separation procedures require the presence of either detergents or organic solvents and a general method has not yet been developed. Electrophoresis methods using modifications of 2D-PAGE protocols with zwitterionic detergents3 as well as * To whom correspondence should be addressed. M.B. (maria.bendz@ cbr.su.se) or P.J. ([email protected]). † CREATE Health, Lund University. ‡ Stockholm University. § Department of Cell & Organism Biology, Lund University. | University of Padova. ⊥ Politecnico di Milano.

5666 Journal of Proteome Research 2009, 8, 5666–5673 Published on Web 10/21/2009

dual detergent systems4-6 and a system for separation membrane protein complexes7 have been explored. Various chromatography methods using extremely hydrophobic solvent systems for reverse phase separation8 or neutral detergents in ion exchange have also been demonstrated.9 However, none of these methods have proven to be as effective as 1D SDS PAGE, though a combination of 1D techniques has been shown to be advantageous.10 The paucity of digestion sites for sequence-specific proteases close to the membrane domains and the general refractory nature toward digestion due to steric hindrance requires the use of strong denaturing agents that are incompatible with protease activity. Lys-C, is active in 1% SDS but generates large peptides which are difficult to separate though the use of high-temperature chromatography has recently been shown to be effective for hydrophobic peptides.11 The first generalized procedure for shotgun membrane protein analysis involved combined Proteinase K and cyanogen bromide digestions.12 Digestion at pH 11 slows down the activity of the protease and generates “ragged” peptides with many overlaps. However, database searching with mass-spectrometry data using non-tryptic peptides is problematic due to the lack of protease cleavage sequence specificity (greatly extending the search space) and the absence of charge localization at either the N- or C-terminus which results in a lack of extended b- or y-ion series and an increase in internal ions. We have shown that this can be circumvented by modifying the peptides with a charge-directing moiety at the N-terminal, such as a nicotinyl group that produces extended b-ion series.13,14 Here we demonstrate an approach to modifying the separation and digestion methods to allow the use of the method for relative quantification of membrane proteins. First we optimized the procedure using standard proteins. We then applied the method to study glucose repression in B. subtilis. Catabolite repression is a regulatory mechanism for global gene expression by which bacteria coordinate the metabolism of carbon and energy sources for maximum efficiency.15 Around 10% of the protein encoding genes are regulated >3 fold by glucose, with repressed genes outnumbering activated genes three to one, at the level of mRNA.16 The negative regulation of transcription of catabolite-repressive genes occurs through the binding of the catabolite control protein (CcpA), which interacts with allosteric effectors such as P-Ser_HPr, that bind to cis-acting catabolite-responsive elements in the DNA.17 Inducer exclusion 10.1021/pr900741t CCC: $40.75

 2009 American Chemical Society

Quantification of Membrane Proteins is independent of catabolite repression mediated by CcpA, which is explained by a specific mechanism to each operon in which a transcriptional repressor is involved.

Materials and Methods Chemicals. Nicotinic acid, N-hydroxysuccinimide, 1-ethyl3-(3-dimethylaminopropyl) carbodiimide hydrochloride, triethylammonium bicarbonate buffer, acetonitrile, acrylamide, N-acryloyl-aminopropanol, urea, Tris, NaOH and iodoacetamide, alpha-cyano-4- hydroxycinnamic acid, N-methyl-piperidine, Protein Assay Kit, and solvents for synthesis and HPLC were from Sigma-Aldrich (Stockholm, Sweden). Dithiothreitol (DTT) and GelCode Blue Stain Reagent were purchased from Pierce Chemical Company (SDS, Falkenberg, Sweden). Sequencegrade-modified trypsin and Proteinase K were purchased from Promega (SDS, Falkenberg, Sweden). D4-Nicotinic acid ethyl ester was purchased from Cambridge Isotope Laboratories (Larodan Fine Chemical AB, Malmoe, Sweden). Synthesis of 1-(Nicotinoyloxy) Succinimide Ester (H4-Nic-NHS). Nicotinic acid (1.23 g, 10 mmol, 1 equiv) and N-hydroxysuccinimide (1.15 g, 10 mmol, 1 equiv) were dissolved in dichloromethane (DCM) (20 mL). 1-Ethyl-3-(3dimethylaminopropyl)carbodiimide hydrochloride (1.92 g, 10 mmol, 1 equiv) was added to the reaction mixture and stirring was continued at room temperature overnight. The solvent was then evaporated on a rotary evaporator and the crude product obtained was dissolved in dichloromethane. The organic phase was extracted three times with a saturated solution of NaCl in water, dried over MgSO4, and then the solvent was evaporated. The crude product was washed with water before being recrystallized from a 30 mL of a 70/30 (v/v) mixture of diethyl ether/methanol at 50 °C. The white precipitate was filtered off and dried under vacuum. The yield was 1.65 g (75%). The purity and structure of the product was confirmed by 1H-NMR (CDCl3, 300 MHz): d ) 2.863 (s, 2 CH2), 7.40-7.50 (m, H5 (ArH)), 8.30-8.40 (dt, H4 (ArH), J ) 1.86, 2.18, 8.1 Hz), 8.80-8.90 (dd, H6 (ArH), J ) 1.87, 4.93 Hz), 9.27 (d, H2 (ArH), J ) 2.18 Hz) ppm. The synthesis of the heavy label was carried exactly as above but substituting the D-4 nicotinic ethyl ester as the precursor. Digestion of Standard Proteins. Solutions of 10 mg/mL of alcohol dehydrogenase (ADH), and bovine serum albumin (BSA) in 100 mM MOPS buffer pH 7 were prepared and 60 µL of each protein solution were placed in Eppendorf tubes. To each tube, 30 µg of trypsin (enzyme/protein 1:20) were added and the mixtures were incubated overnight at 37 °C. Proteinase K digestions were carried out at pH 10 and 12. Twenty-five µL of Proteinase K (10 µg/mL) in 100 mM sodium carbonate buffer was added and after incubation at 37 °C for 3 h a second aliquot of enzyme solution was added and the digestion continued for another 1.5 h. N-Terminal Modification of Standard Protein Peptides. Twelve microliters of the ADH trypsin digest were added to each of two Eppendorf tubes and 2 µL of 200 mM H4-NicNHS in DMF was added to one tube and 2 µL of 200 mM D4-NicNHS in DMF to the other tube. The mixtures were incubated on ice for one hour. Potential side-reactions in the chemical modification of peptides were eliminated by adding 1 µL of 600 µM hydroxylamine in 25 mM Tris buffer pH 8.5 to each tube and incubating for 20 min at room temperature followed by an increase of the pH to 11-12 by adding 6 M NaOH and a final 20 min incubation at room temperature. The tryptic digest of BSA was modified in the same way as the ADH digests. Seven

technical notes microliters of the ADH digest modified with H4-Nic-NHS were mixed with 3 µL of ADH digest modified with D4-Nic-NHS. Ten microliters of the BSA digest modified with H4-Nic-NHS and 10 µL of the BSA digest modified with D4-Nic-NHS were mixed and then added to the ADH mixture. Growth of Bacteria and Isolation of Membranes. Bacillus subtilis 1A1 cells were grown in nutrient sporulation medium with phosphate (NSMP)18 with and without 0.5% (w/v) glucose in 1 L portions in 5 L indented E-flasks. The cultures were grown at 37 °C on a rotary shaker (200 rpm) until they reached early stationary phase. The cells were harvested by centrifugation, 7000× g for 30 min at 4 °C, and washed in 50 mM potassium phosphate pH 8.0. The membrane fraction was isolated from osmotically lysed cells as described before19 and suspended in 20 mM Na-MOPS pH 7.4 and then diluted 20fold in water. The samples were stored at -80 °C until used. Membrane Wash. Isolated B. subtilis membranes were thawed on ice before being centrifuged at 48 000× g for 40 min. The pellet was washed once with ice-cold 10 mM sodium carbonate buffer pH 11, once with ice-cold water and finally two times with 10 mM Tris-HCl buffer pH 7. Between each wash the membrane was pelleted by centrifugation at 48 000× g for 40 min at 4 °C. After the final centrifugation the pellet was suspended in 10 mM Tris-HCl buffer pH 7 and the protein concentration was determined using Protein Assay Kit. 1D-SDS PAGE and Alkylation of the Membrane Proteins. Washed membranes from cells grown with and without glucose, 200 µg protein of each, were added to two Eppendorf tubes. ADH, 10 µg, was added to each tube before the samples were mixed 1:1 with SDS-PAGE sample buffer and heated at 98 °C for 3 min. Proteins in the samples were separated by SDSPAGE according to Laemmli20 using a 15% separation gel and a 5% stacking gel and substituting N-acryloyl-aminopropanol for acrylamide as the matrix monomer. The electrophoresis was run at 25 °C, 25 Amp/gel, until the bromophenol blue dye front had ran off the bottom of the gel. The gel was stained using GelCode Blue Stain Reagent. Each of the lanes was cut into 5 slices. The slices were destained in 50% acetonitrile and 25 mM NH4HCO3, before reduction using 10 mM DTT in 100 mM NH4HCO3 at 55 °C for 1 h. Alkylation of the proteins was done by adding 55 mM iodoacetamide in 100 mM NH4HCO3 to each slice and incubation for 45 min at room temperature in the dark. The slices were then washed once using 100 mM NH4HCO3, then acetonitrile and then two times using water and finally with acetonitrile. In-Gel Digestion of Polypeptides Using Proteinase K. Twenty-five microliters of Proteinase K (10 µg/mL) in 100 mM sodium carbonate buffer pH 12 were added to each washed and dehydrated gel slice. After incubation at 37 °C for 3 h a second aliquot of enzyme solution was added and the digestion continued for another 1.5 h. Peptides were extracted twice from the gel slices by adding 0.1 M HCl in 75% acetonitrile to the slices and incubating at room temperature for 30 min. The samples were dried using a Speedvac before they were dissolved in 15 µL of 200 mM triethyl-ammonium bicarbonate buffer pH 7. Modification of Peptides. Ten percent of each sample was placed in an Eppendorf tubes. Two microliters of prechilled 200 mM H4-NicNHS dissolved in DMF were added to the remaining 90% of each digest of the samples originating from the bacteria grown with the addition of 0.5% glucose and 2 µL of prechilled 200 mM D4-NicNHS dissolved in DMF were added Journal of Proteome Research • Vol. 8, No. 12, 2009 5667

technical notes to the rest of the samples originating from the bacteria grown without 0.5% glucose. To the tubes containing 10% of the samples originating from the bacteria grown with the addition of 0.5% glucose, 0.2 µL of prechilled 200 mM D4-NicNHS dissolved in DMF was added, and to the tubes containing 10% of the samples originating from the bacteria grown without 0.5% glucose, 0.2 µL of prechilled 200 mM H4NicNHS dissolved in DMF was added. The reactions were carried out for one hour on ice. Then the “10%” and “90%” samples were pooled. Side-reaction products with Ser, Thr, and Tyr residues were eliminated by adding 1 µL of 600 µM hydroxylamine in 25 mM Tris buffer pH 8.5 to each sample and then incubating for 20 min at room temperature. The pH in each sample was then increased to 11-12 by adding 6 M NaOH and the incubation was carried on for 20 min at room temperature. The H4-Nic-NHS and D4Nic-NHS samples were pooled and then the pH was decreased to 2 by the addition of 12 M HCl. Mass Spectrometry. All samples were analyzed on a Q-tof Ultima API (Waters, Manchester, UK) coupled to a Waters CapLC HPLC. The auto sampler injected 6 µL of sample and the peptides were trapped on a precolumn (C18, 300 µm × 5 mm, 5 µm, 100 Å, LC-Packings), and separated on a reversed phase analytical column (Atlantis, C18, 75 µm × 150 mm, 3 µm, 100 Å, Waters) with the flow rate set to 200 nL/min. Solvent A consisted of 2% acetonitrile, 98% water, with 0.1% formic acid. Solvent B consisted of 90% acetonitrile, 10% water, and 0.1% formic acid. The HPLC method started at 5% B for 18 min, was then raised from 5 to 80% B over 57 min, from 80 to 100% B over 1 min, held at 100% B for 25 min before returning from 80 to 5% B in 1 min and re-equilibrating at 5% B for 15 min. The total run time was 115 min. The mixture of modified standard ADH and BSA peptides was diluted 10-fold before being injected while the membrane protein peptide samples were injected directly. Analysis of HPLC-MS Data. Qtof raw data files were converted to mzXML using wolf (http://sashimi.sourceforge.net). The data from the membrane samples were optionally cleaned before further analysis, as described previously21 to obtain better peak detection. Peak features were extracted using msInspect (build 658422). The output peak lists, which include maximum peak intensities, charge states and apex retention times, were analyzed using a plug-in to Proteios Software Environment (msInspect PairFinder, freely available at http://www.proteios.org). Using the plug-in, each peak list was searched for multiply charged peptide pairs that differed 4 Da (between the calculated parent pairs) with a tolerance of (0.05 Da, and with peak retention times that differed less than 30 s. Peaks that differed in intensity by at least 20% were put into an include list for MS/MS acquisition. Singly charged peaks were also included if they had a mass of at least 550 and an intensity larger than 100. Three include lists were generated for each run; one in which the lower mass peaks were more abundant, one in which the high mass peaks of the pairs were more intense and one for multiply charged singlet ions and for singly charged peaks with a mass of at least 550 and an intensity of at least 100. Optimising Collision Energy for Modified Peptides. To find the optimal collision energy for the modified peptides, 10 µL of the ADH digest modified with H4-Nic-NHS, were diluted 1:10 in 0.1% formic acid. The samples were analyzed as described in the previous section. Dynamic Data Acquisition, (DDA) was used for automatic MS/MS over a mass range m/z from 400 to 5668

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Bendz et al. 1600 for MS and from 50 to 1800 for MS/MS. The instrument was set to analyze +1, +2, and +3 ions during all runs. The instrument analyzed the sample twice, with high collision energy in the first run and lower collision energy in the second run. The collision energy decision tree approach is given in Supplementary Table 2, Supporting Information. MS/MS Analysis. All samples analyzed using MS-HPLC were analyzed again in MS/MS mode. The samples containing a mixture of N-terminal modified standard peptides were injected in total three times using the different inclusion lists. The membrane samples were analyzed six times, as each inclusion list were analyzed twice using either high or low collision energy. The instrument was set to analyze +1, +2, and +3 ions during all runs. Database Analysis. All HPLC-MS/MS files were processed using ProteinLynx V2.2 and the pkl-files originating from the same inclusion list but with different collision energy were merged. The files were analyzed using Mascot version 2.2.03 and with enzyme set to None, peptide mass tolerance: ( 0.1 Da, fragment mass tolerance: ( 0.1 Da against the B. subtilis strain 168 database downloaded from the Subtilis database (www.genolist.pasteur.fr). Carbamidomethyl and H4-/D4-NicNHS N-terminal modifications, were set as fixed modifications, while H4/D4-Nic-NHS (Lys) and oxidation (Met) modification was set to be variable modifications. A Mascot score of 41 was used which corresponds to a false positive cutoff rate for proteins of 95%.

Results General Strategy. The approach used in this work to identify membrane-bound proteins in B. subtilis and determine their relative amounts depending on the growth medium is shown schematically in Figure 1. Isolated cell membrane fractions were washed with alkaline buffer, to remove loosely associated proteins and then subjected to one-dimensional SDS-PAGE. The electrophoresis step results in fractionation and denaturation of the proteins, which increases the accessibility to proteases during the following enzymatic digestion. Samples to be compared (membranes from cells grown with and without 0.5% glucose in the medium) were run next to each other to make sure that they were cut out similarly from the gel. The procedure was done in duplicate to check reproducibility and yeast Alcohol dehydrogenase was added to each membrane sample prior to SDS-PAGE to act as an internal standard. Optimisation of Polypeptide Digestion. During the development of the method we used in-gel digestions of Alcohol dehydrogenase, with Proteinase K. Although this protease is supposed to cut polypeptides unspecifically analyses of the resulting peptides showed that certain peptides prevailed. It therefore seems as if Proteinase K has some preferences as to where to cut in the amino acid sequence. Others groups have also reported this.23 In our published method,14 we used pH 11 of the enzyme buffer during protein digestion. The high pH was used to control the digestion in order to get peptides of a length suitable for MS analysis. During in-gel digestion we suspected that carbon dioxide from the air was lowering the actual pH of the digestion buffer resulting in shorter peptides than expected. To obtain longer peptides and hence more reliable MS results, we increased the pH of the enzyme buffer to 12 for in-gel digestions. The trade off was increased hydrolysis of the gel itself increasing the MS background. Using the alkali stable matrix based on the N-acryloyl-aminopropanol

Quantification of Membrane Proteins

Figure 1. Schematic representation of the method. The proteins in the membrane fractions are separated by 1D SDS-PAGE. The proteins are digested in-gel using Proteinase K at pH 12. The resulting peptides derived from growth condition 1 are modified using 90% light label and those from growth condition 2, with 90% heavy. The samples are pooled and analyzed using RPHPLC-MS. The raw files are filtered for noise, and peak detection is carried out with msInspect and the output MZXML files analyzed by Pairfinder, which identifies peptide pairs and makes an inclusion list of those peptides changing in relative intensity between light and heavy. The peptides in the inclusion lists are then analyzed by RP-HPLC-MS/MS.

monomer ameliorated this.24,25 The increase in pH also decreased the exopeptidase activity and the number of peptide “ladders” (data not shown). Database Searching with Peptides from Nonspecific Protease Digestions. We previously showed that the N-terminal modification NicNHS dramatically increases the number of peptides matched and the scores obtained when using 1+/2+ ion selection as compared to nonmodified peptides.14 In this work, we focused on determining the relative amounts of those that were found affected by the growth condition. From the RP-HPLC MS/MS files where the Pairfinder software had been used to create an include list for the Q-TOF, Mascot was able to identify 41 protein differently expressed under the two growth conditions (shown in cartoon form in Figure 2 with details in Supplementary Tables 2 and 3, Supporting Information). Among those, 19 appeared in the RP-HPLC-MS file as doublets while the other appeared as singlets. As we modified all peptides 90:10 both isotopic labels, all peptides are expected to appear as doublets. However, due to the noise level, sometimes one of the peptides in the pair was not visible above background. All proteins identified in Mascot were manually evaluated before being accepted as an identification. The MS/MS spec-

technical notes trum for each peptide from these identified proteins were manually analyzed. The identified proteins derived from doublet peptides were accepted if (i) the protein was identified in both runs and with a consistent regulation between all peptides or (ii) when the protein only appeared in one of the samples it should have a good Mascot score and a consistent regulation between all peptides. If the peptides from the identified protein were singlets, the identification was accepted if the protein appeared as a singlet in both samples and at least one peptide from each sample had a good Mascot score. All identified proteins were analyzed using the membrane protein prediction programs, TMHMM 2.0 and SOSUI. We also used a new prediction program Octopus26 giving the correct topology for over 90% of the membrane proteins compared to for example TMHMM 2.0 that estimates the correct topology for about 70% of the proteins. The prediction programs showed that several of the identified proteins are membrane proteins with transmembrane spanning helices. In order to obtain more information about the proteins, we also used the UNIPROT entry annotations. In some cases we found that the proteins were peripheral proteins being parts of membrane proteins complexes. Identified Membrane Proteins Whose Expression Is Affected by Glucose in the Medium. Four proteins of the ATP synthase complex (AtpA, AtpD, AtpE, and AtpB) were identified as being repressed during growth with glucose in the medium. The four proteins are part of the membrane peripheral part (F1) of the enzyme. Repression of the ATP synthase complex by glucose has also been reported for Bacillus licheniformis which is a bacterium very similar to B. subtilis.27 Also, the flavoprotein subunit (SdhA) of succinate dehydrogenase was found to be repressed several fold by glucose, which is consistent with published data for B. subtilis.28 The App and the Opp systems are ABC-type transporters for oligopeptides in B. subtilis. The Opp system, but not the App system, can transport tripeptides.29 Two proteins of the Opp system (OppA, located on the periplasmic side and OppF, and one of the two ATP binding proteins located on the cytoplasmic side) were found to be down-regulated in the presence of glucose.30 The reproducibility of the relative quantification is illustrated in Table 1, which shows the regulation of the individual peptides from OppA. The OppD ATP binding protein, belonging to the same transport complex, was found to be upregulated during growth with glucose. The Opp transport system has been thoroughly studied in both Salmonella typhimuruium31-34 and B. subtilis.29,30 The PTS system glucosespecific EIICBA component (PtsG) was found to be upregulated in growth with glucose as carbon source. The protein is a polytopic membrane protein predicted by Octopus to have twelve trans-membrane helices and is involved in uptake of sugars.35 Menaquinol oxidase, cytochrome aa3, is the major respiratory oxidase in B. subtilis. The enzyme consists of four polypeptides36 and two of these, subunit 1 (QoxB) and subunit 2 (QoxA), were identified and found to be up-regulated during growth with glucose. Flagellin (hag) was found in isolated membranes and upregulated in the sample grown with glucose. It has been suggested that the flagellar structure is assembled from the cell membrane outward and that additions are made to the nascent organelle with proteins that are transported through the core of the organelle and accumulate at the outer tip.37,38 Studies have shown that hag gene transcription is entirely an exponential-phase phenomenon. Transcription occurs while glucose Journal of Proteome Research • Vol. 8, No. 12, 2009 5669

technical notes

Bendz et al.

Figure 2. Cartoon diagram of the regulated membrane proteins identified in B. subtilis. Known transport functions are shown in the figure. Dashed arrows indicate putative functions. Proteins up-regulated during growth with glucose as carbon source are shown in green, those up-regulated during growth with amino acids are yellow and protein/protein complexes not identified but interacting with an identified protein are in gray. Table 1. Regulation of the Individual Peptides in the Oligopeptide-Binding Protein (OppA)a peptide identified

relative expression sample A

relative expression sample B

TFEGL SLHPGLA LDDVAVK DWAGMPL KEYLEK HVEPIAGVY LHVEPIAGVY DFEYA GWLGDF EQIPAMAAVP GQLPTESLPTLK

0.61 0.75 0.45 0.65 0.64 0.59 0.41 0.36 0.89 0.37 0.40

N/A N/A 0.48 0.37 N/A 0.34 0.36 0.67 0.64 0.79 0.22

a Isotope ratios (heavy/light) for the samples grown with and without glucose are shown for sample A and sample B. Sample A gives a mean value of 0.56 (SD 0.17) and sample B 0.48 (SD 0.19). The combined mean and SD are 0.53 and 0.18.

is present and being utilized, and it ceases when this substance is exhausted. This suggests that either the presence or the utilization of the primary energy or carbon source of growth is somehow a requirement for σD-dependent gene expression.39 The findings indicate that flagellin synthesis in B. subtilis is not subject to glucose catabolite repression as in Escherichia coli.40

Discussion The procedure we have developed involves the use of isolated membrane preparations that are extensively washed 5670

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at high pH to remove contaminating proteins prior to 1D SDS-PAGE. Paired samples are run in adjacent gel lanes before slicing, cysteine alkylation and in-gel digestion using Proteinase K at pH 12 (Figure 1). The pH is critical. At around pH 9, the digestion proceeds very rapidly yielding very small (3-5 amino acids) peptides that are uninformative for protein identification. At pH 10, the proteinase is not so active, and produces medium (15 amino acids) sized peptides that have very ragged ends due to extensive exoprotease activity. We therefore raised the pH to 12 and could obtain peptides that were much less ragged. This removed the first obstacle toward a method that can be applied for quantification. We had seen that the highly denaturing conditions of SDS/PAGE gave the best digestion results for all enzymes investigated. The high pH used with Proteinase K however resulted in hydrolysis of the polymeric gel matrix producing extensive non-peptide peaks in the HPLC-MS analysis and frequent blockage of the nano-HPLC systems. We therefore used a pH stable 3-acryloylamino-1-propanol instead of the usual acrylamide matrix monomer.41 The matrix is very hydrophilic and greatly increased peptide recovery. In addition, no matrix hydrolysis was observed and the high pH eliminated most of the exopeptidase activity. One of the main problems in analyzing membrane proteins is defining what a membrane protein is. There are four main types of membrane proteins, those with transmembrane spanning regions, transient membrane proteins (that usually possess a targeting TM domain that is subsequently removed), covalently attached proteins (via fatty acids or glycosylphosphatidylinositol anchors) and mem-

Quantification of Membrane Proteins brane associated proteins (bound noncovalently to the membrane or to TM proteins). The availability of complete bacterial genome sequences allows proteome-wide predictions of exported proteins. It is computationally difficult to distinguish between (Sec-type) signal peptides that direct protein export and lipoprotein signal peptides or aminoterminal membrane anchors that cause protein retention in the membrane. A recent study42 was aimed at improving methods for the prediction of protein retention in the bacterial cytoplasmic membrane. This was based on using sets of membraneattached and extracellular proteins from B. subtilis that were recently identified through proteomics approaches. The results showed that three classes of membrane-attached proteins could be distinguished. Two classes include 43 lipoproteins and 48 proteins with an amino-terminal transmembrane segment, respectively. Remarkably, a third class includes 31 proteins that remain membrane-retained despite the presence of typical Sec-type signal peptides with consensus signal peptidase recognition sites. The results were based on previously reported proteome analyses in which the true fraction of membrane proteins was not rigorously controlled. In a recent study describing the global topology analysis of the E. coli inner membrane proteome43 the authors established the periplasmic or cytoplasmic locations of the C termini for 601 inner membrane proteins. By constraining a topology prediction algorithm with this data, they derived high-quality topology models for the 601 proteins, providing a firm foundation for future functional studies of this and other membrane proteomes. One of the first reports on a proteome level analysis of bacterial membrane proteins was published by Bunai.38 They analyzed ABC transporter solute-binding proteins of B. subtilis using a washed cell membrane fraction that was insoluble in 134 mM sulphobetaine. The proteins remaining were then extracted from the membranes using mixtures of detergents in a stepwise manner and subjected to 2-D polyacrylamide gel electrophoresis. After electroblotting, digestion and MALDI peptide fingerprinting, 637 proteins were identified. Of these, 256 were predicted to be membrane proteins, 101 were lipoproteins or secretory proteins and the remainder were soluble proteins. In a similar approach for the analysis of Chlorobium tepidum membrane proteins,44 different methods were employed for the enrichment of membrane proteins prior to analysis with 2-DE. Isolated membranes were solubilized with Triton X-100 and from the supernatant 58 proteins were identified. The use of SDS for protein solubilization, combined with acetone precipitation, resulted in an improved 2-DE pattern and the total number of identified proteins was increased to 117. More than 100 of these proteins are not predicted to be membrane proteins. This further confirms that 2D-PAGE is probably not a suitable method for analyzing membrane proteins. A large-scale study of the proteome of growing cells of B. subtilis was based on using standard 2D gel systems together with supplementary zoom gels (pI 5.5-6.7, 5-6, 4.5-5.5, and 4-5) and resulted in identification of 693 proteins.45 In addition to the cytosolic neutral and alkaline proteins, 130 membrane proteins were found by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation in combination with electrospray ionization-tandem mass spectrometry (ESI-MS/MS) techniques. Of these pro-

technical notes teins, we find that only about 30 are membrane proteins as predicted by the consensus of the three algorithms. More recent reports have described alternative approaches to analyzing intrinsic membrane proteins using combinations of alternative electrophoresis techniques. One strategy involved washing the membranes with 2 M NaBr to disassociate soluble proteins and membrane-associated organelles.46 These proteins are then separated as membrane protein complexes using 1D Blue-native polyacrylamide gel electrophoresis after solubilization with n-dodecyl-beta-D-maltoside (DDM) followed by a second dimension of Tricine-SDSPAGE. By this approach 143 different proteins were identified of which 50% appeared to be TM proteins. A similar study on E. coli-derived native outer membrane vesicles yielded 141 proteins.47 Finally a novel non-gel based method was described using a chemical approach of labeling the exposed proteins on the bacterial surface in E. coli with dansyl chloride labeling.48 This was followed by tryptic digestion and two-dimensional tandem mass spectrometry. The proteins identified could be grouped into three major families: outer membrane (29 proteins), lipoproteins (6 proteins), and transmembrane (43 proteins) families. Thirty proteins were confirmed as TM proteins as defined by a unanimous consensus by the predictors. The remaining known proteins were found to be parts of known membrane complexes (Figure 2). Four proteins of the ATP synthase complex (AtpA, B, D, and E) were identified as being repressed during growth with glucose in the medium as has been shown for B. licheniformis.27 Menaquinol oxidase, cytochrome aa3, is the major respiratory oxidase in B. subtilis. The enzyme consists of four polypeptides and two of these, subunit 1 (QoxB) and subunit 2 (QoxA), were identified and found to be up-regulated during growth with glucose as was the PTS system glucose-specific EIICBA component (PtsG), a twelve TM protein. The Opp system is ABC-type transporter for oligopeptides, transporting tripeptides.29 Two proteins of the Opp system (OppA, located on the periplasmic side and OppF, and one of the two ATP binding proteins located on the cytoplasmic side) were found to be down-regulated in the presence of glucose. The reproducibility of the peptide ratios for OppA is shown in Table 1. This method is a robust and reproducible way to obtain accurate quantitative data on changes in membrane protein composition and is applicable to any system. As we have previously shown, the number of membrane proteins (especially those with small extra-membrane domains) identified is dramatically increased with this approach. The increase coverage combined with quantification will help unravel at least one part of the invisible proteome. Abbreviations: Nic, nicotinic acid; NHS, N-hydroxysuccinimide ester; H4, light form; D4, heavy (deuterated) isotopomer; ADH, alcohol dehydrogenase; NSMP, nutrient sporulation medium with phosphate; MOPS, 3-(N-morpholino)propanesulfonic acid; DMF, dimethylformamide; TM, transmembrane.

Acknowledgment. This work was supported by grants from the Gothenburg Research School in Functional Genomics (M.B. and M.C.M.); the Knut and Alice Wallenberg Foundation (P.J.); grant 621-2007-6094 from the Swedish Research Council (L.H.); and from the Swedish Strategic Research Council to CREATE Health (P.J.). S.C. was supported by a grant from the BLANCEFLOR Foundation Journal of Proteome Research • Vol. 8, No. 12, 2009 5671

technical notes Boncompagni-Ludovisi. M.B. performed the digestion and labeling experiments and participated in writing the manuscript. M.C.M. performed the bacterial growth and membrane isolation and purification. G.A. performed the manual interpretation of MS/MS data. Å.W. carried out the MS analysis. R.S. developed the high pH gel method and suppression of exopeptidase activity. S.C. applied the noise filtering methods. F.L. carried out the comparative MS data analysis. L.H. designed and supervised the bacteriological work. P.J. initiated and designed the study and contributed to writing the manuscript. All authors discussed the results and commented on the manuscript.

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